Methods of attaching biological compounds to solid supports using triazine

ABSTRACT

Disclosed are methods of attaching biologically active compounds to a solid surface, comprising modifying the solid surface using triazine chloride and attaching the biologically active compound to the triazine moiety.

RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 12/364,286, now U.S. Pat. No. 7,863,058 filed Feb. 2, 2009, byZhao et al., and entitled “METHODS OF ATTACHING BIOLOGICAL COMPOUNDS TOSOLID SUPPORTS USING TRIAZINE” the entire disclosure of which isincorporated by reference herein, including any drawings, which is adivisional of Ser. No. 11/271,433 filed Nov. 10, 2005, now U.S. Pat. No.7,504,499 by Zhao et al., and entitled “METHODS OF ATTACHING BIOLOGICALCOMPOUNDS TO SOLID SUPPORTS USING TRIAZINE” the entire disclosure ofwhich is incorporated by reference herein, including any drawings, whichis a continuation-in-part of U.S. application Ser. No. 10/739,959, filedon Dec. 17, 2003, now U.S. Pat. No. 7,259,258 by Kozlov et al., andentitled “METHODS OF ATTACHING BIOLOGICAL COMPOUNDS TO SOLID SUPPORTSUSING TRIAZINE,” the entire disclosure of which is incorporated byreference herein, including any drawings.

FIELD OF THE INVENTION

The present disclosure relates to the field of attachment ofbiologically active compounds, such as oligonucleotides and peptides, tosolid surfaces.

BACKGROUND OF THE INVENTION

High-throughput analysis of oligonucleotides and peptides requires theimmobilization of these compounds to solid surfaces. Various techniquesexist in the art today for this purpose. However, these methods arecumbersome and achieve their task at considerable time and cost to theuser. In addition, with many of the current methods, it is not possibleto introduce different sequences onto the same solid surface at the sametime.

SUMMARY OF THE INVENTION

Disclosed are methods of attaching biologically active compounds to asolid surface, comprising modifying the solid surface using triazinechloride and attaching the biologically active compound to the triazinemoiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the two-step reaction sequence for attachingtwo oligonucleotides to a bead.

FIG. 2 is a schematic of the one-pot reaction sequence for attaching twooligonucleotides to a bead.

FIG. 3 is a schematic of a reaction sequence for attachingoligonucleotides to a bead.

FIG. 4 is another schematic of the two-step reaction sequence forattaching two oligonucleotides to a bead.

FIG. 5 is another schematic of the one-pot reaction sequence forattaching two oligonucleotides to a bead.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the first aspect, the present disclosure describes a compound ofFormula I

wherein

-   -   X¹ and X² are each independently selected from the group        consisting of chloro, NH—NH₂, NH—N═CH-E-T-L-D,        -   wherein        -   E is a bond or an electron withdrawing group;        -   T is selected from a bond, —C(O)—, —C(O)NH—, —NHC(O)—, an            oxygen atom, a sulfur atom, NH, —S(O)—, —SO₂—, or alkyl            substituted with one or more substituents selected from the            group consisting of dialkylamine, NO₂, CN, SO₃H, COOH, CHO,            alkoxy, and halogen;        -   L is a linker selected from the group consisting of a bond,            —(CH₂)_(p)—, —(CH₂)_(p)—O—, —(CH₂)_(p)—C(O)—,            —(CH₂)_(p)—C(O)NH—, —(CH₂)_(p)—NHC(O)—, —(CH₂)_(p)—S(O)—,            —(CH₂—CH₂—O)_(p)—, and —(CH₂)_(p)—S(O)₂—; and        -   D is a biologically active polymer;    -   Q is a solid surface;    -   V is selected from a bond, NH, CO, NHCO, C(O)NH, (CH₂)_(p),        sulfur, and oxygen, or a combination thereof; and    -   p is an integer greater than or equal to zero.

In certain embodiments, V is NH.

In some embodiments, E comprises an aryl, heteroaryl, heterocyclyl,alkyl, or cycloalkyl group.

In certain embodiments, E is a bond and T is a bond. Accordingly, inthese embodiments, X₁ and X₂ are each independently NH—N═CH-E-D, where Eand D are as defined above.

As used herein, the term “alkyl” refers to an aliphatic hydrocarbongroup. The alkyl moiety can be a “saturated alkyl” group, which meansthat it does not contain any alkene or alkyne moieties. The alkyl moietycan also be an “unsaturated alkyl” moiety, which means that it containsat least one alkene or alkyne moiety. An “alkene” moiety refers to agroup consisting of at least two carbon atoms and at least onecarbon-carbon double bond, and an “alkyne” moiety refers to a groupconsisting of at least two carbon atoms and at least one carbon-carbontriple bond. The alkyl moiety, whether saturated or unsaturated, can bebranched, straight chain, or cyclic.

The alkyl group can have 1 to 20 carbon atoms (whenever it appearsherein, a numerical range such as “1 to 20” refers independently to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group can also be a mediumsize alkyl having 1 to 10 carbon atoms. The alkyl group could also be alower alkyl having 1 to 5 carbon atoms. The alkyl group of the compoundsof the preferred embodiments can be designated as “C₁-_(n) alkyl” orsimilar designations, where n is an integer value. By way of exampleonly, “C₁-₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Accordingly, alkyl or other moieties disclosedherein can alternatively have straight chain or branched structures.

The alkyl group can be substituted or unsubstituted. When substituted,the substituent group(s) is(are) one or more group(s) individually andindependently selected from cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio,arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato,isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino,including mono- and di-substituted amino groups, and the protectedderivatives thereof. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and the like. Wherever asubstituent is described as being “optionally substituted” thatsubstitutent can be substituted with one of the above substituents.

In the present context, the term “cycloalkyl” is intended to coverthree-, four-, five-, six-, seven-, or eight-or more membered ringscomprising carbon atoms only. A cycloalkyl can optionally contain one ormore unsaturated bonds situated in such a way, however, that an aromaticπ-electron system does not arise. Some examples of “cycloalkyl” are thecarbocycles cyclopropane, cyclobutane, cyclopentane, cyclopentene,cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene,1,4-cyclohexadiene, cycloheptane, or cycloheptene.

The term “heterocyclyl” is intended to mean three-, four-, five-, six-,seven-, and eight- or more membered rings wherein carbon atoms togetherwith from 1 to 3 heteroatoms constitute the ring. A heterocyclyl canoptionally contain one or more unsaturated bonds situated in such a way,however, that an aromatic π-electron system does not arise. Theheteroatoms are independently selected from oxygen, sulfur, andnitrogen.

A heterocyclyl can further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclicthioimides, cyclic carbamates, and the like.

Heterocyclyl rings can optionally also be fused to aryl rings, such thatthe definition includes bicyclic structures. Typically such fusedheterocyclyl groups share one bond with an optionally substitutedbenzene ring. Examples of benzo-fused heterocyclyl groups include, butare not limited to, benzimidazolidinone, tetrahydroquinoline, andmethylenedioxybenzene ring structures.

Some examples of “heterocyclyls” include, but are not limited to,tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin,1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane,1,4-oxathiin, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine,maleimide, succinimide, barbituric acid, thiobarbituric acid,dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane,hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran,pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline,pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3-dioxolane,1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, and1,3-oxathiolane. Binding to the heterocycle can be at the position of aheteroatom or via a carbon atom of the heterocycle, or, for benzo-fusedderivatives, via a carbon of the benzenoid ring.

In the present context the term “aryl” is intended to mean a carbocyclicaromatic ring or ring system. Moreover, the tend “aryl” includes fusedring systems wherein at least two aryl rings, or at least one aryl andat least one C₃₋₈-cycloalkyl share at least one chemical bond. Someexamples of “aryl” rings include optionally substituted phenyl,naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl,indenyl, and indanyl. The term “aryl” relates to aromatic, including,for example, benzenoid groups, connected via one of the ring-formingcarbon atoms, and optionally carrying one or more substituents selectedfrom, but not limited to, heterocyclyl, heteroaryl, halo, hydroxy,amino, cyano, nitro, alkylamido, acyl, C₁₋₆ alkoxy, C₁₋₆ alkyl, C₁₋₆hydroxyalkyl, C₁₋₆ aminoalkyl, C₁₋₆ alkylamino, alkylsulfenyl,alkylsulfenyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl. The arylgroup can be substituted at the para and/or meta positions. In otherembodiments, the aryl group can be substituted at the ortho position.Representative examples of aryl groups include, but are not limited to,phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl,3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl,3-methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl,hydroxymethylphenyl, trifluoromethylphenyl, alkoxyphenyl,4-morpholin-4-ylphenyl, 4-pyrrolidin-1-ylphenyl, 4-pyrazolylphenyl,4-triazolylphenyl, and 4-(2-oxopyrrolidin-1-yl)phenyl.

In the present context, the term “heteroaryl” is intended to mean aheterocyclic aromatic group where one or more carbon atoms in anaromatic ring have been replaced with one or more heteroatoms selectedfrom the group comprising nitrogen, sulfur, phosphorus, and oxygen.

Furthermore, in the present context, the term “heteroaryl” comprisesfused ring systems wherein at least one heteroaryl ring shares at leastone chemical bond with another carbocyclic ring. Thus, heteroaryl caninclude, for example, fused ring systems wherein at least one aryl ringand at least one heteroaryl ring, at least two heteroaryl rings, atleast one heteroaryl ring and at least one heterocyclyl ring, or atleast one heteroaryl ring and at least one cycloalkyl ring share atleast one chemical bond.

The term “heteroaryl” is understood to relate to aromatic, C₃₋₈ cyclicgroups further containing one oxygen or sulfur atom or up to fournitrogen atoms, or a combination of one oxygen or sulfur atom with up totwo nitrogen atoms, and their substituted as well as benzo- andpyrido-fused derivatives, for example, connected via one of thering-forming carbon atoms. Heteroaryl groups can carry one or moresubstituents, selected from halo, hydroxy, amino, cyano, nitro,alkylamido, acyl, C₁₋₆-alkoxy, C₁₋₆-alkyl, C₁₋₆-hydroxyalkyl,C₁₋₆-aminoalkyl, C₁₋₆-alkylamino, alkylsulfenyl, alkylsulfinyl,alkylsulfonyl, sulfamoyl, or trifluoromethyl. In some embodiments,heteroaryl groups can be five- and six-membered aromatic heterocyclicsystems carrying 0, 1, or 2 substituents, which can be the same as ordifferent from one another, selected from the list above. Representativeexamples of heteroaryl groups include, but are not limited to,unsubstituted and mono- or di-substituted derivatives of furan,benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole,oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole,isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole,quionoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine,furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole,triazole, benzotriazole, pteridine, phenoxazole, oxadiazole,benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, andquinoxaline. In some embodiments, the substituents are halo, hydroxy,cyano, O—C₁₋₆-alkyl, C₁₋₆-alkyl, hydroxy-C₁₋₆-alkyl, andamino-C₁₋₆-alkyl.

In certain embodiments, E is selected from the group consisting of

where the phenyl ring may be optionally substituted with one or moresubstituents selected, without limitation, from the group consisting ofdialkylamine, NO₂, CN, SO₃H, COOH, CHO, alkoxy, and halogen.

In other embodiments, E is selected from the group consisting of —C(O)—,—S(O)—, —SO₂—, and alkyl optionally substituted with one or moresubstituents selected from the group consisting of dialkylamine, NO₂,CN, SO₃H, COOH, CHO, alkoxy, and halogen.

D can be a bioactive polymer as exemplified above, however D can be anyof a variety of molecules including, for example, an organic molecule ora biologically active molecule or both. In certain embodiments, D isselected from the group consisting of a small organic molecule, apolymer, a macromolecule, an oligonucleotide, and a polypeptide. Incertain embodiments, the oligonucleotide is selected from RNA, DNA, RNAhaving one or more non-naturally occurring bases, DNA having one or morenon-naturally occurring bases. In other embodiments D is a polypeptideor a polypeptide having one or more non-naturally occurring amino acids.In particular embodiments, D can possess no known biological activity,the biological activity of D can be under investigation or the primaryinterest in D is not its biological activity.

As used herein, the terms “nucleic acid,” “polynucleotide,” or“oligonucleotide,” and other grammatical equivalents, refer to at leasttwo nucleotides covalently linked together. A nucleic acid of thepreferred embodiments will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that can have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989)), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C&E News Jun. 2, 1997 page 35. In additionnucleic acids include “locked nucleic acids” (LNAs) such as thosedescribed in Koshkin et al., J. Am. Chem. Soc. 120: 13252-3 (1998). LNAis a novel type of nucleic acid analog that contains a 2′-O, 4′-Cmethylene bridge. This bridge restricts the flexibility of theribofuranose ring and locks the structure into a rigid bicyclicformation, conferring enhanced hybridization performance and exceptionalbiostability. Other useful nucleic acids are described in Khudyakov etal., Artificial DNA Methods and Applications CRC Press, NY 2003. All ofthese references are hereby expressly incorporated by reference.

The oligonucleotides useful in the molecules and methods describedherein can have modified bases. Modified bases can comprise a moietyselected from, but not limited to, an aldehyde, a heterocyclic ring, acarbocyclic ring, an aryl ring, or a heteroaryl ring. Examples ofmodified bases include, but are not limited to, optionally substitutedpurines, optionally substituted pyrimidines, optionally substitutedphenyl groups, optionally substituted naphthyl groups, optionallysubstituted pyridyl groups, and the like.

The oligonucleotides can have modified sugars. Exemplary sugarmodifications for the oligonucleotide include sugars with six-memberedrings. Other useful sugar modifications are described in Khudyakov etal., Artificial DNA Methods and Applications CRC Press, NY 2003.

Thus, the term “oligonucleotide” refers to an oligomer or polymer ofnucleotides or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. The term“oligonucleotide” can include nucleotides having a ribose or adeoxyribose. Similarly, the term “nucleotide” is used as recognized inthe art to include natural bases, and modified bases well known in theart. Such bases are generally located at the 1′ position of a sugarmoiety. Nucleotide generally comprises a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety. An oligonucleotide, polynucleotide ornucleic acid within the scope of the present disclosure can include, forexample, at least 5, 7, 10, 12, 15, 17, 20, 25, 30, 35, 40, 50, 60, 70or 80 or more nucleotides. These molecules can also be shorter having,for example, at most 80, 70, 60, 50, 40, 30, 20, 10, or 5 nucleotides.Nucleic acid molecules described herein can also have a length betweenany of these upper and lower limits.

Other molecules such as organic molecules having at least one carbonatom can be used in the molecules or methods described herein. “Smallorganic molecules” are molecules comprising at least one carbon atom andthat have a molecular weight of less than 500 g/mol. Small organicmolecules may be naturally occurring or be synthesized in a laboratory.In the context of the present disclosure, small organic molecules do notinclude polypeptides having more than two amino acids oroligonucleotides having more than 2 nucleotides. However, small organicmolecules can comprise 1 or 2 amino acids, or 1 or 2 nucleotides. Incertain embodiments, the small organic molecules are biologicallyactive. In these embodiments, the small organic molecules may modulatethe activity of an enzyme or may bind to a receptor in, or on thesurface of, a cell. Certain of these molecules can be used aspharmaceuticals. In other embodiments, the small organic moleculespossess no known biological activity, or that the primary interest inthe molecule is not for its biological activity. An organic moleculeuseful for use in the methods or compounds described herein, such as asmall organic molecule, can be a non-polymeric molecule such as anon-polypeptide or non-polynucleotide molecule.

“Polypeptides” or “peptides” refer to molecules that comprise two ormore amino acids linked together through an amide or a peptide bond. Theamino acids forming a polypeptide or peptide may be naturally occurringamino acids or non-naturally occurring amino acids. Naturally occurringamino acids are those that are typically L-amino acids and have α-sidechains found in nature. Non-naturally occurring amino acids are aminoacids that may be either an L or D isomer and have α-side chains notfound in nature. Non-naturally occurring amino acids also include the Disomer of the naturally occurring amino acids. A polypeptide or peptideas described herein can include, for example, at least 5, 7, 10, 12, 15,17, 20, 25, 30, 35, 40, 50, 60, 70 or 80 or more amino acids. Thesemolecules can also be shorter having, for example, at most 80, 70, 60,50, 40, 30, 20, 10, or 5 amino acids. Polypeptode molecules of thepresent disclosure can also have a length between any of these upper andlower limits.

In particular embodiments D can be a library or mixture of compounds. Asdemonstrated below in Example 4, a hydrazine treated solid phasesubstrate can be reacted with a mixture of polynucleotides to yieldbeads derivatized with a mixture of polynulceotides. Other mixtures canbe used, such as a library of molecules. Libraries of molecules usefulfor the methods and compositions described herein can be obtained using,for example, well known methods of combinatorial synthesis built fromany of a variety of building blocks. Exemplary building blocks andreagents are nucleic acids, amino acids, other organic acids, aldehydes,alcohols, and so forth, as well as bifunctional compounds, such as thosegiven in Krchnak et al., 1996, “Synthetic library techniques: Subjective(biased and generic) thoughts and views,” Molecular Diversity, 1:193-216. A library that can be used in accordance with the presentdisclosure can also be obtained using recombinant methods withbiological cells or cellular components.

The term “solid surface” is defined as a material having a rigid orsemi-rigid surface to which a compound described herein can be attachedor upon which they can be synthesized. As set forth above, Q can be asolid surface. A solid surface can be found on or in a solid-phasesupport or substrate. In certain embodiments, Q is selected from thegroup consisting of resin, microbead, glass, controlled pore glass(CPG), polymer support, membrane, paper, plastic, plastic tube ortablet, plastic bead, glass bead, slide, ceramic, silicon chip,multi-well plate, nylon membrane, fiber optic, and PVDF membrane. Incertain embodiments, the solid surface Q may comprise functional groups,such as —NH₂, —OH, —SH, —COOH, etc., on its surface. In otherembodiments, the surface of Q may be functionalized prior to thereaction with a triazine molecule. Other materials that can be used inaccordance with the present disclosure include, but are not limited to,polypropylene, polyethylene, polybutylene, polyurethanes, nylon, metals,and other suitable materials. In some embodiments, the material ismalleable or pliable, as discussed below. A solid surface can be aparticle, for example, made of cross-linked starch, dextrans, cellulose,proteins, organic polymers including styrene polymers includingpolystyrene and methylstyrene as well as other styrene co-polymers,plastics, glass, ceramics, acrylic polymers, magnetically responsivematerials, colloids, thoriasol, carbon graphite, titanium dioxide,nylon, latex, or TEFLON®. “Microsphere Detection Guide” from BangsLaboratories, Fishers, Inc., hereby incorporated by reference in itsentirety, is a helpful guide. Further exemplary substrates within thescope of the present disclosure include, for example, those described inUS Application Publication No. 02/0102578 and U.S. Pat. No. 6,429,027,both of which are incorporated herein by reference in their entirety,including any drawings.

In accordance with certain embodiments, one or more solid-phasesupports, such as particles, can be securely attached to a secondsupport or substrate thus being useful in high-throughput synthesis(“HTS”) apparatus. In particular embodiments Q can be located at or in aparticular feature of a solid-phase support substrate such as a well ordepression. A solid phase support substrate can include one or morereaction vessels or wells. For example, Q can be a bead or otherparticle located in a well or depression or Q can be located at or in afeature of a substrate by virtue of being a part of the substrateitself. One or more solid-phase supports can be disposed within eachwell and affixed to the substrate. Such configuration allows syntheticreactions to be performed within one or more wells of the substrate. Inparticular, liquid reagents can be added to each well, reacted withineach well, and then the residual liquid can removed from each well viacentrifugation or aspiration. In one embodiment, the substrate is amicrotiter plate that includes a plurality of wells disposed in arraysincluding, but not limited to, the rectangular array of wells disposedon a 96-well or 384-well microtiter plate.

As noted above, one or more solid-phase supports can be affixed to asubstrate within each well. In some embodiments, the solid-phasesupports are pressed and embedded into the substrate while the substrateis heated to an elevated temperature approximating the melting point ofthe substrate. As the substrate cools, the solid-phase supports arepermanently bonded to the substrate, which bond is capable ofwithstanding the centrifugal forces often found in centrifugalseparation. Alternatively, the solid phase support can be adhesivelybonded to the internal diameter of the well utilizing a thermoplasticmaterial having a relatively low melting point (i.e., lower than that ofthe substrate), a two-part epoxy, and/or other suitable means.

In particular embodiments, Q can be a chemical moiety or linker.Accordingly, the present disclosure includes soluble compounds havingstructures similar to those exemplified herein for solid substrates withthe exception that the solid substrate can be replaced by any of avariety of known chemical moieties. The present disclosure furtherprovides methods for adding a moiety to an aldehyde- or amine-containingcompound or for linking together two aldehyde- or amine-containingcompounds. By way of example, the methods disclosed herein can be usedto synthesize a soluble compound of Formula VIII, X, XIII, XIV, or XVIIIin which L is a linker. Q can be a linker selected from the groupconsisting of a bond, the moieties identified as L above, a polypeptide,a polynucleotide and any of a variety of other linkers known in the art.Thus, the present disclosure provides polypeptides attached topolynucleotides via triazine moieties, polypeptides attached topolypeptides via triazine moieties, polynucleotides attached topolynucleotides via triazine moieties and other combinations of polymersand/or organic molecules exemplified herein with respect to modificationof solid surfaces. Further examples include, without limitation,attachment of antibodies to polynulceotides, attachment of enzymes topolynucleotides, attachment of organic molecules (for example in acombinatorial synthesis) to polynucleotides, and attachment ofpolysaccharides to polypeptides such as enzymes or antibodies.

In certain embodiments, p is an integer less than 50. In otherembodiments, p is less than 30, while in other embodiments, p is lessthan 20. In some embodiments p is less than 10. In certain embodiments,p is greater than 2, while in other embodiments, p is greater than 5.

Certain embodiments of the present disclosure relate to a compound ofFormula I in which E is

In other embodiments, T is —C(O)NH—. In still other embodiments, L is abond, while in some other embodiments L is —(CH₂)_(p)—NHC(O)—. In someof these embodiments, p is 6. In yet other embodiments D is DNA, whilein other embodiments D is a peptide.

In certain embodiments, the compound of Formula I is selected from thegroup consisting of

In another aspect, the present disclosure is related to the compound ofFormula XX

-   -   wherein    -   X¹ and X² are each independently selected from the group        consisting of chloro, NH—NH₂, NH—N(X⁹)(X¹⁰), Z-D, NH—N═CH—Z-D,        NH—N═Z-D, NH—NH—Z-D and NH—Z-D,    -   wherein    -   Z can be a bond or a linker; and    -   D is a biologically active polymer;    -   X⁹ and X¹⁰ are each independently selected from the group        consisting of alkyl, alkoxy, aryl, and heteroaryl, or X⁹ and X¹⁰        taken together along with the nitrogen atom to which they are        attached form a five- or six-membered alicyclic, heterocyclic,        aromatic, or heteroaromatic ring;    -   V is selected from a bond, SiO₃, NH, CO, NHCO, C(O)NH,        (CH₂)_(p), sulfur, and oxygen, or a combination thereof;    -   p is an integer greater than or equal to zero; and    -   Q is a solid surface.

In some embodiments, X¹ and X² are both chloro. In other embodiments, X¹and X² are both NHNH₂. In other embodiments, one of X¹ and X² is chlorowhile the other of X¹ and X² is NHNH₂. In further embodiments, one of X¹and X² is NHNH₂ while the other of X¹ and X² is NH—N═CH—Z-D.

In certain embodiments, D is selected from the group consisting of RNA,DNA, a polypeptide, RNA having one or more non-naturally occurringbases, DNA having one or more non-naturally occurring bases, and apolypeptide having one or more non-naturally occurring amino acids.

In some embodiments, D is DNA.

In some embodiments, V is NH, while in other embodiments, V is—NH—(CH₂)_(p)—Si(O)₃ where p is an integer greater than or equal tozero, such as p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or a numbergreater than 10.

In some embodiments, Q is selected from the group consisting of resin,microbead, glass, controlled pore glass (CPG), polymer support,membrane, paper, plastic, plastic tube or tablet, plastic bead, glassbead, slide, ceramic, silicon chip, multi-well plate, nylon membrane,and PVDF membrane.

The coupling between Z and D is broad and covers many differentconnections. In some embodiments, when D is a polynucleotide, Z caninclude the sugar, the phosphate and the base of the first nucleotide ofthe polynucleotide molecule. In other embodiments, Z can include onlythe sugar and the phosphate of the first nucleotide of thepolynucleotide molecule. In still other embodiments, Z can include onlythe sugar of the first nucleotide of the polynucleotide molecule. In yetstill other embodiments Z may include none of the sugar, the phosphateor the base of the first nucleotide of the polynucleotide molecule.

In another aspect, the present disclosure is related to a compound ofFormula XVIII,

wherein

-   -   X⁵, X⁶, X⁷, and X⁸, are each independently selected from the        group consisting of chloro, NH—NH₂, NH—N═CH-E-T-L-D,        -   wherein        -   each E is independently a bond or an electron withdrawing            group;        -   each T is independently selected from a bond, —C(O)—,            —C(O)NH—, —NHC(O)—, an oxygen atom, a sulfur atom, NH,            —S(O)—, —SO₂—, or alkyl substituted with one or more            substituents selected from the group consisting of            dialkylamine, NO₂, CN, SO₃H, COOH, CHO, alkoxy, and halogen;        -   each L is independently a linker selected from the group            consisting of a bond, —(CH₂)_(p)—, —(CH₂)_(p)—O—,            —(CH₂CH₂O)_(p), —(CH₂)_(p)—C(O)—, —(CH₂)_(p)—C(O)NH—,            —(CH₂)_(p)—NHC(O)—, —(CH₂)_(p)—S(O)—, and —(CH₂)_(p)—S(O)₂—;            and        -   each D is independently a biologically active polymer;    -   Q is a solid surface;    -   V¹ and V² are each independently selected from a bond, NH, CO,        NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination        thereof; and    -   p is an integer greater than or equal to zero.

In another aspect, the present disclosure is related to the compound ofFormula XXVI

-   -   where    -   X⁵, X⁶, X⁷, and X⁸, are each independently selected from the        group consisting of chloro, NH—NH₂, NH—N(X⁹)(X¹⁰), Z-D,        NH—N═CH—Z-D, NH—N═Z-D, NH—NH—Z-D and NH—Z-D,    -   where,        -   each Z is a bond or a linker; and        -   each D is independently a biologically active polymer;        -   X⁹ and X¹⁰ are each independently selected from the group            consisting of alkyl, alkoxy, aryl, and heteroaryl, or X⁹ and            X¹⁰ taken together along with the nitrogen atom to which            they are attached form a five- or six-membered alicyclic,            heterocyclic, aromatic, or heteroaromatic ring;    -   Q is a solid surface; and    -   V¹ and V² are each independently selected from a bond, SiO₃, NH,        CO, NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a        combination thereof; and        -   p is an integer greater than or equal to zero.    -   Z can be any kind of chemical linker that can connect Formula XX        or XXVI to the biologically active polymer D. Examples of Z        include, but are not limited to, a bond, the moieties identified        as L above, a polypeptide, a polynucleotide or any of a variety        of other chemical linkers known in the art.

In some of the embodiments, all of the substituents D may be the same,while in other embodiments, all of the substituents D may be different.For example, a plurality of compounds disclosed herein having the samesubstituents D can be useful in a method of synthesizing a desiredquantity of substituent D. An exemplary use of a plurality of compoundsdisclosed herein having different substituents D is the production of anarray for use in a diagnostic application. Accordingly binding of atarget analyte to a particular substituent D in an array of differentsubstituents can identify a property of the target analyte or the samplefrom which it is derived. In yet other embodiments, some of thesubstituents D are the same while others are different. Embodimentsinclude those in which each D is a peptide, while in other embodiments,each D is an oligonucleotide. When all of the substituents D arepeptides or all are oligonucleotides, the sequence of the peptide or theoligonucleotide may be the same for all of the substituents D, or may bedifferent for all of the substituents D, or may be the same for some ofthe substituents and different for other substituents. Accordingly,disclosed herein are oligonucleotide or peptide arrays.

In particular embodiments, a compound disclosed herein or a populationof these compounds can include two or more types of molecules assubstituents D. For example, the substituents D can include a polymerand a second molecule. In embodiments, where a population of moleculesis used, each molecule can be associated with a polymer having a uniquesequence such that the polymer encodes the identity of the molecule. Forexample, a unique oligonucleotide can be bound to each array locationwhere a particular member of a population of second molecules is boundsuch that identification of the oligonucleotide sequence identifies thestructure or other property of the molecule at the array location. Thecompounds and methods described herein can be used in known methods ofencoding or decoding such as those described in WO 03/002979, WO01/46675, and WO 99/67641, all of which are incorporated by referenceherein in their entirety, including any drawings.

Thus, in some embodiments X⁵ is peptide, while in other embodiments X⁶is peptide. In certain embodiments X⁷ is peptide, while in still otherembodiments X⁸ is peptide. Similarly, in some embodiments X⁵ is anoligonucleotide, while in other embodiments X⁶ is an oligonucleotide. Incertain embodiments X⁷ is an oligonucleotide, while in still otherembodiments X⁸ is an oligonucleotide.

In another aspect, the present disclosure is related to a compound ofthe Formula XXI:

-   -   where    -   X₁ is selected from the group consisting of chloro and NH—NH₂;        and    -   D is a biologically active polymer;    -   V is selected from a bond, SiO₃, NH, CO, NHCO, C(O)NH,        (CH₂)_(p), sulfur, and oxygen, or a combination thereof;        -   p is an integer greater than or equal to zero; and    -   Q is a solid surface.

In another aspect, the present disclosure is related to a compound ofthe Formula XXVII:

where

-   -   X₁ is selected from the group consisting of chloro and NH—NH₂;        and    -   D₁ and D₂ are the same or different biologically active        polymers;    -   Q is a solid surface;    -   V is selected from the group consisting of a bond, SiO₃, NH, CO,        NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination        thereof; and    -   p is an integer greater than or equal to zero.

In another aspect disclosed herein is a method of synthesizing acompound of Formula XXVII

wherein

-   -   D1 and D2 are the same or different biologically active        polymers, Q is a solid surface, X¹ is chloro or —NHNH₂, and    -   V is selected from the group consisting of a bond, SiO₃, NH, CO,        NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination        thereof; and    -   p is an integer greater than or equal to zero;        comprising:    -   silanating a solid surface with a silanating compound or        providing a solid surface having at least one group reactive        towards cyanuric chloride;    -   reacting the solid surface with cyanuric chloride and hydrazine        to obtain a compound of Formula XXII

-   -   wherein Q is a solid surface and V is selected from the group        consisting of a bond, SiO₃, NH, CO, NHCO, C(O)NH, (CH₂)_(p),        sulfur, and oxygen, or a combination thereof; and    -   p is an integer greater than or equal to zero; and    -   reacting the compound of Formula XXII with one or more aldehyde        modified biologically active polymers to obtain a compound of        Formula XXVII.

Some solid surfaces already exhibit groups on the surface, which canreact with a cyanuric chloride compound. For example, glass has —OHgroups on the surface, which is reactive towards cyanuric chloride. Thehydroxyl group can substitute the chloro to form an ether linkage. Othersolid surfaces do not naturally have a group that is reactive towardscyanuric chloride. These solid surfaces can be modified by reacting thesolid surface with a reagent that attaches a group that is reactivetowards cyanuric chloride on the surface.

In some embodiments, the group that is reactive towards cyanuricchloride is a nucleophile. In certain embodiments, the nucleophile isselected from the group consisting of hydrazine, amine, thiol, andhydroxyl.

In yet another aspect, the present disclosure is related to a compoundwherein the compound of Formulas XX is selected from the groupconsisting of:

-   -   wherein Q is a solid surface.

In another aspect, disclosed herein is a method of synthesizing acompound of Formula I

wherein

-   -   X¹ is Cl and X² is NH—NH₂ or X¹ and X² are both NH—NH₂ and Q is        a solid surface, and V is selected from a bond, NH, CO, NHCO,        C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination thereof;    -   comprising    -   reacting a solid surface having —NH₂, —COH, —COOH, —NHCOH,        —NHCOOH, —C(O)NH₂, —(CH₂)_(p)CH₃, —SH, or —OH, or a combination        thereof, on its surface with triazine chloride to obtain a        compound of Formula III

-   -   reacting the compound of Formula III with hydrazine to obtain a        compound of Formula I.

The solid surfaces can have any of a variety of different reactivegroups, including, but not limited to, —NH₂, —OH, —SH, or —NHNH₂. Someof the conditions for reacting a solid surface having —NH₂ with triazinechloride are provided in Example 1 below. Similar solvents andconditions can be used for solid surfaces having other reactive groups.Those skilled in the art will know or be able to determine appropriatesolvents and conditions based on known properties of the particularreactive group being used.

Examples of solvents for reactions involving hydrazine, triazines orother compounds disclosed herein include, but are not limited to,acetonitrile, acetone, n-butyl acetate, carbon tetrachloride,chlorobenzene, chloroform, cyclohexane, cyclopentane, dimethylacetamide, dimethyl formamide, dimethyl sulfoxide, dioxane, ethylacetate, ethyl ether, ethylene dichloride, heptane, hexadecane,iso-octane, methyl t-butyl ether, methyl ethyl ketone, methyl isoamylketone, methyl isobutyl ketone, methyl n-propyl ketone, methylenechloride, n-methylpyrrolidone, pentane, petroleum ether, pyridine,tetrahydrofuran, toluene, 1,2,4-trichlorobenzene, trichloroethylene,trichlorotrifluoroethane, o-xylene and mixtures thereof.

A compound of Formula I is useful for derivatizing a surface Q with asecond compound having an aldehyde. The aldehyde can react with thedistal amine of an NH—NH₂ moiety to form an imine linkage between thecompound of Formula I and the second compound. Thus, in cases where Q ofFormula I is a solid surface the present disclosure provides a methodfor immobilizing a compound having a reactive aldehyde moiety. Inparticular embodiments, aldehydes used in a methods described herein forthe synthesis of compounds described herein will have an electronwithdrawing group including, but not limited to

where the phenyl ring may be optionally substituted with one or moresubstituents selected, without limitation, from the group consisting ofdialkylamine, NO₂, CN, SO₃H, COOH, CHO, alkoxy, and halogen.

In another aspect, the present disclosure is related to a method ofsynthesizing a compound of Formula XXII

-   -   wherein Q is a solid surface and V is selected from a bond,        SiO₃, NH, CO, NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a        combination thereof; and    -   p is an integer greater than or equal to zero;    -   comprising: silanating a solid surface with a silanating        compound,    -   reacting the silanated solid surface with cyanuric chloride and        hydrazine to obtain a compound of Formula XXII.

As used herein, the terms “silanating” and “silanizing” refers broadlyto attaching any silicon-containing compound to another compound,surface or substrate. Examples of silanating compounds include, but arenot limited to aminopropyltrimethoxysilane,γ-aminopropyltriethoxysilane, dimethylchlorosilane, and the like. Acompound thus reacted is referred to as a “silanated” or “silanized”compound.

In another aspect, disclosed herein is a method of synthesizing acompound of Formula II

wherein

-   -   D is a biologically active polymer, Q is a solid surface, X³ is        chloro or —NHNH₂, and V is selected from a bond, NH, CO, NHCO,        C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination thereof;    -   comprising    -   reacting a solid surface having —NH₂, —COH, —COOH, —NHCOH,        —NHCOOH, —C(O)NH₂, —(CH₂)_(p)CH₃, —SH, or —OH, or a combination        thereof; on its surface with triazine chloride to obtain a        compound of Formula III

-   -   reacting the compound of Formula III with hydrazine and a        compound of Formula V

to obtain a compound of Formula II.

In certain embodiments, reacting the compound of Formula III withhydrazine results in the formation of a compound of Formula IV, while inother embodiments, this reaction results in compound of Formula VII.

Thus, in some embodiments hydrazine replaces both chlorides in thecompound of Formula III, whereas in other embodiments, hydrazinereplaces only one of the chloride substituents. For example, one molarequivalent or less of hydrazine can be reacted with a compound ofFormula III in order to form a compound of Formula VII. Alternatively, atwo or more fold molar excess of hydrazine can be reacted with acompound of Formula III in order to form a compound of Formula IV.

In another aspect, the present disclosure refers to a method ofsynthesizing a compound of Formula XXI

where

-   -   D is a biologically active polymer;    -   X₁ is chloro or —NHNH₂, and V is selected from a bond, SiO₃, NH,        CO, NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a        combination thereof;        -   p is an integer greater than or equal to zero; and    -   Q is a solid surface;        comprising:    -   silanating a solid surface with a silanating compound,    -   reacting the silanated solid surface with cyanuric chloride and        hydrazine to obtain a compound of Formula XXII

-   -   reacting the compound of Formula XXII with an aldehyde modified        biologically active polymer to obtain a compound of Formula XXI.

In some embodiments, the aldehyde modified biologically active polymercan be selected from the group consisting of RNA, DNA, a polypeptide,RNA having one or more non-naturally occurring bases, DNA having one ormore non-naturally occurring bases, and a polypeptide having one or morenon-naturally occurring amino acids.

As used herein “aldehyde modified biologically active polymer” refers toa biologically active polymer which has an aldehyde on one or more ofits substituents. This includes any location on such a polymer and thepolymer can be in any state of polymerization or can exist as a singlemonomer of the polymer. In some embodiments, the aldehyde modifiedbiologically active polymer is modified on the 5′ end of a DNA or RNAmolecule. In other embodiments, the aldehyde modified biologicallyactive polymer is modified on the 3′ end of the DNA or RNA molecule. Insome embodiments, the aldehyde modified biologically active polymer ismodified on the C-terminal of a polypeptide. In other embodiments, thealdehyde modified biologically active polymer is modified on theN-terminal of a polypeptide. In some embodiments, the aldehyde modifiedbiologically active polymer is modified on the base of the firstnucleotide of a polynucleotide.

In certain embodiments, the modified base on the aldehyde modifiedbiologically active polymer has the following structure:

wherein D is a biologically active polymer.

In other embodiments, the aldehyde modified biologically active polymerhas the structure of the compound of Formula XXIII:

In these embodiments, the compound of Formula XXIII reacts with thehydrazinyl moiety on the compound of Formula XXII, thereby forming asubstituted morpholin, which has the result of attaching thebiologically active polymer to the solid surface. The reaction isdepicted in the following scheme.

In some embodiments, the silanating compound can be R—Si(OR′)₃,R₁(R₂)—Si—(OR′)₂ or R₁(R₂)(R₃)—Si—(OR′). In certain embodiments, the R,R₁, R₂, and R₃ group of the silanating compound can each independentlycontain any or a combination of —NH—, —(CH₂)_(p)—, —C(O)— and —O—,wherein p is an integer greater than or equal to zero. In someembodiments, R, R₁, R₂, and R₃ are each independently selected fromoptionally substituted alkyl, optionally substituted aryl, optionallysubstituted alkoxy, and optionally substituted aryloxy. R′ can behydrogen, optionally substituted alkyl, optionally substituted aryl,optionally substituted alkoxy, or optionally substituted aryloxy. Insome embodiments, the silanating compound isaminopropyltrimethoxysilane.

Thus, in some embodiments, a solid surface, such as a silica bead, glassbead, resin, or any other solid surface disclosed herein, is silanizedby a silanating compound. The silanating compound preferably contains analkylamino group attached to the silicon atom such that, followingsilanization, an alkylamino group, having a primary amino group, isattached to the solid surface. The modified solid surface is thenreacted with cyanuric chloride to obtain a compound of Formula III,where V is an aminoalkylsilane group. The cyanuric activated solidsurface is then reacted with hydrazine to replace the remaining twochloro substituents on the cyanuric group with hydrazinyl moieties.Aldehyde modified oligonucleotides are then reacted with modified solidsurface. The aldehyde moiety of the oligonucleotide molecule reacts withthe hydrazinyl moiety on the solid surface, thereby attaching theoligonucleotide to the solid surface and immobilizing it.

Another aspect of the present disclosure refers to a method ofsynthesizing a compound of the Formula XXV

where

-   -   Q is a solid surface;    -   V₁ and V₂ are selected from a bond, NH, CO, NHCO, C(O)NH,        (CH₂)_(p), sulfur, SiO₃ and oxygen, or a combination thereof;    -   X³ and X⁴ are each independently chloro or —NH—NH₂, and p is an        integer greater than or equal to zero;        comprising:    -   silanating a solid surface with a silanating compound, and    -   reacting the silanated solid surface with cyanuric chloride and        hydrazine to obtain the compound of Formula XXV.

Another aspect of the present disclosure refers to a method of obtaininga solid surface to which a plurality of biologically active polymers areattached, and having a structure of Formula XXIV,

where

-   -   D₁ is a biologically active polymer having a first primary        sequence;    -   D₂ is a biologically active polymer having a second primary        sequence;    -   where the first primary sequence and the second primary sequence        may be the same or different;    -   Q is a solid surface;    -   X³ and X⁴ are each independently chloro or —NH—NH₂;    -   n and m are each independently 1 or an integer greater than 1;        and    -   V¹ and V² are selected from a bond, NH, CO, NHCO, C(O)NH,        (CH₂)_(p), sulfur, SiO₃ and oxygen, or a combination thereof;        and    -   p is an integer greater than or equal to zero        comprising:        providing a compound of Formula XXV

-   -   and reacting a compound of Formula XXV with one or more aldehyde        modified biologically active polymers to obtain the compound of        Formula XXIV.

In yet another aspect, disclosed herein is a method of synthesizing acompound of Formula II

wherein

-   -   D is a biologically active polymer, Q is a solid surface, X³ is        chloro or —NHNH₂, and V is selected from a bond, NH, CO, NHCO,        C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination thereof;    -   comprising    -   providing a compound of Formula III

-   -   reacting the compound of Formula III with hydrazine and a        compound of Formula V

to obtain a compound of Formula II.

In certain embodiments, V is NH.

The methods of synthesis described above relate to single attachmentchemistry, i.e., methods of synthesis where only a single type ofsubstituent D is to be attached to the solid surface. There are timeswhen it is desirable to attach two or more different types ofsubstituent D to a single solid surface.

Thus, in another aspect, disclosed herein is a method of obtaining asolid surface to which a plurality of biologically active polymers areattached, and having a structure of Formula XIII,

-   -   wherein L is a linker selected from the group consisting of a        bond, —(CH₂)_(p)—, —(CH₂)_(p)—O—, —(CH₂)_(p)—C(O)—,        —(CH₂)_(p)—C(O)NH—, —(CH₂)_(p)—NHC(O)—, —(CH₂)_(p)—S(O)—,        —(CH₂—CH₂—O)_(p)—, and —(CH₂)_(p)—S(O)₂—, wherein p is an        integer greater than or equal to zero; and    -   D₁ is a biologically active polymer having a first primary        sequence;    -   D₂ is a biologically active polymer having a second primary        sequence;        -   wherein said first primary sequence and said second primary            sequence may be the same or different;    -   Q is a solid surface;    -   X³ and X⁴ are each independently chloro or —NH—NH₂;    -   V¹ and V² are each independently selected from a bond, NH, CO,        NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination        thereof; and    -   n and m are each independently 1 or an integer greater than 1;    -   comprising:    -   reacting a solid surface having —NH₂, —CHO, —COOH, —NHCOH,        —NHCOOH, —C(O)NH₂, —(CH₂)_(p)CH₃, —SH, or —OH, or a combination        thereof, on its surface with triazine chloride to obtain a        compound of Formula VIII

-   -   reacting the compound of Formula VIII with a compound of Formula        IX        H₂N-L-D₁  (IX)    -   to obtain a compound of Formula X

-   -   reacting the compound of Formula X with hydrazine and a compound        of Formula XII

to obtain a compound of Formula XIII.

An amine compound disclosed herein can be obtained commercially orsynthesized using methods known in the art. For example, a nucleic acidcan be derivatized to include an amino group using methods described inNucleic Acid Research, 13: 2399-2412 (1985); Nucleic Acid Research, 14:7985-7994 (1986); Tetrahedron Letters, 27:3991-3994 (1986); Nucleic AcidResearch, 7:3131-3139 (1987); Nucleic Acid Research, 15: 6209-6224(1987); Tetrahedron Letters, 28:2611-2614 (1987); or Nucleic AcidResearch, 17: 7179-7186 (1989). Such methods can be used withconventional equipment or, if desired, high throughput methods canemploy robotic devices. In embodiments using robotics or other equipmentfor manipulations of the reactions, solvents for the reactions can bereplaced with those that are compatible with the equipment such asacetonitrile or other inert solvent.

Reactions between amine compounds or aldehyde compounds and triazinescan be carried out in the presence of salts if desired. Exemplary saltsthat are useful include, but are not limited to sodium chloride, lithiumchloride, magnesium chloride, potassium chloride, sodium sulfate,lithium sulfate, magnesium sulfate, potassium sulfate, or salts ofphosphate or acetate. Salts can be present in concentrations of 0.01,0.05, 0.1, 0.5, 1, 2, 3, 4, or 5 M or higher.

A “primary sequence” is the sequence of amino acids in a polypeptide ora sequence of nucleotides in an oligonucleotide.

In certain embodiments, D₁ or D₂ is an organic molecule, such as a smallorganic molecule, instead of a biologically active polymer. In someembodiments, both D₁ and D₂ are organic molecules. In these embodiments,D₁ and D₂ can be two different molecules.

In certain embodiments, V¹ and V² are each NH. In some embodiments, D₁and D₂ are both peptides, while in other embodiments D₁ and D₂ are botholigonucleotides. In yet other embodiments, D₁ is a peptide and D₂ is anoligonucleotide. As set forth previously herein, D₁ or D₂ canindependently be a polymer, such as a polypeptide or polynucleotide, oran organic molecule or any of a variety of combinations thereof.

In another aspect, the present disclosure relates to a method ofobtaining a solid surface to which a plurality of biologically activepolymers are attached, and having a structure of Formula XIII,

-   -   wherein L is a linker selected from the group consisting of a        bond, —(CH₂)_(p)—, —(CH₂)_(p)—O—, —(CH₂)_(p)—C(O)—,        —(CH₂)_(p)—C(O)NH—, —(CH₂)_(p)—NHC(O)—, —(CH₂)_(p)—S(O)—,        —(CH₂—CH₂—O)_(p)—, and —(CH₂)_(p)—S(O)₂—, wherein p is an        integer greater than or equal to zero; and    -   D₁ is a biologically active polymer having a first primary        sequence;    -   D₂ is a biologically active polymer having a second primary        sequence;        -   wherein said first primary sequence and said second primary            sequence may be the same or different;    -   Q is a solid surface;    -   X³ and X⁴ are each independently chloro or —NH—NH₂;    -   V¹ and V² are each independently selected from a bond, NH, CO,        NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination        thereof; and    -   n and m are each independently 1 or an integer greater than 1;    -   comprising:    -   providing a compound of Formula VIII

-   -   reacting the compound of Formula VIII with a compound of Formula        IX        H₂N-L-D₁  (IX)    -   to obtain a compound of Formula X

-   -   reacting the compound of Formula X with hydrazine and a compound        of Formula XII

to obtain a compound of Formula XIII.

An embodiment relating to the above methods is depicted in FIG. 1. Asshown in the figure, a bead to which at least two molecules of triazinechloride are attached reacts with an amine-modified oligonucleotide(Oligonucleotide 1). The resulting complex is then reacted withhydrazine, whereby the chloride substituents on the triazine moietiesare replaced by hydrazine. The product is then reacted with analdehyde-modified oligonucleotide (Oligonucleotide 2). Theamine-modified oligonucleotide selectively reacts with achloro-substituted triazine, while the aldehyde-modified oligonucleotideselectively reacts with the hydrazine-substituted triazine. Thus, in twosimple steps, two different oligonucleotides are attached to the solidsurface.

While FIG. 1 and the above discussion relate to oligonucleotides, itshould be understood that the above procedure can be easily modified toattach other types of compounds, such as peptides or small organicmolecules, to triazine-modified solid surfaces. Thus, generally, in thefirst step, an amine-modified compound is reacted with the triazinechloride-modified solid surface. The resulting complex is then reactedwith hydrazine, whereby the chloride substituents on the triazinemoieties are replaced by hydrazine. The product is then reacted with analdehyde-modified compound to obtain the final product. Those skilled inthe art will recognize that a compound being reacted in the first stepshould not contain reactive amines other than the amine desired toparticipate in the reaction with the triazine chloride-modified solidsurface. Similarly, a compound being reacted in the second step shouldnot contain a reactive aldehyde group other than the one desired toparticipate in the reaction with the hydrazine treated, triazinechloride-modified solid surface. In embodiments where amine or aldehydereactive groups are present in a compound used in the presentdisclosure, but are not desired to participate in a particular reaction,the reactive groups can be selectively blocked from participating in thereactions using a blocking group or a protecting group. Those skilled inthe art will know or be able to determine appropriate protecting groupchemistry based on the properties of the compound used. For example,common protected forms of aldehydes and amines can be found in Greeneand Wuts Protective Groups in Organic Synthesis; John Wiley and Sons:New York, 1991.

In a further aspect, disclosed is a method of obtaining a solid surfaceto which a plurality of biologically active polymers are attached, andhaving a structure of Formula XIV,

-   -   wherein L is a linker selected from the group consisting of a        bond, —(CH₂)_(p)—, —(CH₂)_(p)—O—, —(CH₂)_(p)—C(O)—,        —(CH₂)_(p)—C(O)NH—, —(CH₂)_(p)—NHC(O)—, —(CH₂)_(p)—S(O)—,        —(CH₂—CH₂—O)_(p)—, and —(CH₂)_(p)—S(O)₂—, wherein p is an        integer greater than or equal to zero; and    -   D₁ is a biologically active polymer having a first primary        sequence;    -   D₂ is a biologically active polymer having a second primary        sequence;        -   wherein said first primary sequence and said second primary            sequence may be the same or different;    -   Q is a solid surface;    -   X³ and X⁴ are each independently chloro or —NH—NH₂;    -   V¹ and V² are each independently selected from a bond, NH, CO,        NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or a combination        thereof; and    -   n and m are each independently 1 or an integer greater than 1;    -   comprising:    -   reacting a solid surface having —NH₂, —COH, —COON, —NHCOH,        —NHCOOH, —C(O)NH₂, —(CH₂)_(p)CH₃, —SH, or —OH, or a combination        thereof, on its surface with triazine chloride to obtain a        compound of Formula VIII

-   -   reacting a compound of Formula VIII with hydrazine and compounds        of Formulae XVI and XVII

to obtain a compound of Formula XIV.

Further aspects of the present disclosure include a method of obtaininga solid surface to which a plurality of biologically active polymers areattached, and having a structure of Formula XIV,

-   -   wherein L is a linker selected from the group consisting of a        bond, —(CH₂)_(p)—, —(CH₂)_(p)—O—, —(CH₂)_(p)—C(O)—,        —(CH₂)_(p)—C(O)NH—, —(CH₂)_(p)—NHC(O)—, —(CH₂)_(p)—S(O)—,        —(CH₂—CH₂—O)_(p)—, and —(CH₂)_(p)—S(O)₂—, wherein p is an        integer greater than or equal to zero; and    -   D₁ is a biologically active polymer having a first primary        sequence;    -   D₂ is a biologically active polymer having a second primary        sequence;        -   wherein said first primary sequence and said second primary            sequence may be the same or different;    -   Q is a solid surface;    -   X³ and X⁴ are each independently chloro or —NH—NH₂; and    -   n and m are each independently 1 or an integer greater than 1;    -   comprising:    -   providing a compound of Formula VIII

-   -   reacting a compound of Formula VIII with hydrazine and compounds        of Formulae XVI and XVII

to obtain a compound of Formula XIV.

An embodiment of the above methods is depicted in FIG. 2. As shown inFIG. 2, first a solid surface is modified with triazine chloride. Theresulting complex is then reacted with hydrazine, whereupon the chloridesubstituents on the triazine are replaced with hydrazine. Then a mixtureof at least two aldehyde-modified oligonucleotides is reacted with themodified solid surface. While in some embodiments both hydrazinesubstituents react with the oligonucleotides, in other embodiments onlyone of the hydrazine moieties reacts with an oligonucleotide.

While FIG. 2 and the above discussion relate only to oligonucleotides,it should be understood that the above procedure can be easily modifiedto attach other types of compounds, such as peptides or small organicmolecules, to triazine-modified solid surfaces. Thus, generally, in thefirst step, an triazine chloride-modified solid surface is reacted withhydrazine, whereby the chloride substituents on the triazine moietiesare replaced by hydrazine. The product is then reacted with a mixture ofaldehyde-modified compounds to obtain the final product.

EXAMPLES

The examples below are not limiting and are only illustrative of some ofthe embodiments disclosed herein.

Example 1 Hydrazine Bead Preparation

Cyanuric chloride activated beads were prepared as follows. 10 g ofamino-modified beads were suspended with 20 μL of acetonitrile. Then 0.8mL of N,N-diisopropylethylamine (DiPEA) and 100 mg of cyanuric chloridewere added to the bead suspension. The reaction was mixed by vortexingand shaking at room temperature for 2 hrs. After the reaction, the beadsolution was centrifuged and the supernatant removed. The beads werethen washed with 100 μL of acetonitrile three times.

The beads were washed six times with DMF. For this and the followingwashes solvent was added to reach 10% bead solid content solution. Thebeads were shaken with 2% hydrazine solution in DMF overnight at roomtemperature using 10% bead solid content. Beads were then washed sixtimes with DMF. The hydrazine beads were stored as a 10% bead solidcontent solution in DMF at room temperature or used directly. Inaddition to DMF, this chemistry works well with other solvents, such asacetonitrile and ethanol. The hydrazine solution was found to be stableover long storage periods including up to at least 100 hours.

Example 2 Immobilization of Aldehyde Group Containing Oligonucleotideson Hydrazine Beads

Beads were prepared as described in Example 1. Beads were washed onetime with 100 mM Na-citrate buffer, 3 M NaCl, pH 5.0. An aldehydecontaining oligonucleotide called sequence 13 (25mer, 2-4 nmols ofoligonucleotide per one mg of beads) was added to the washed beads in100 mM Na-citrate buffer, 3 M NaCl, pH 5.0. The mixture was shakenovernight at room temperature using 10% bead solid content. The beadswere then washed six times with water, followed by three washes withethanol. Beads were stored as a 10% bead solid content solution inethanol.

Example 3 Sequential Immobilization of Amino Group ContainingOligonucleotides and Aldehyde Group Using Hydrazine Beads

Amino group containing oligonucleotides were reacted at roomtemperature, overnight, in aqueous buffer with cyanuric chloridemodified beads, prepared as described in Example 1. Beads were washedwith aqueous buffer and then ethanol. Following attachment of aminogroup containing oligonucleotides to the beads, aldehyde groupcontaining oligonucleotides were immobilized as described in Example I.

Activity of the beads was evaluated using a hybridization assay in whichfluorescently labeled oligonucleotide probes, having sequencescomplementary to the immobilized oligonucleotides, were hybridized tothe beads and detected in a fluorescent activated cell sorter (FACS).When sequential two-attachment chemistry is used, the hybridizationintensities for the first (9mer, attached using amino group containingoligonucleotide) and second attachment (13mer, attached using aldehydegroup containing oligonucleotide) were affected by the hydrazineconcentrations and reaction times of the hydrazine treatment.Hybridization efficiency increased as incubation time increased from 5minutes to overnight. The FACS hybridization assay was used to determinethe effects of different concentrations of hydrazine on hybridization ofprobes to the 9mer and 13mer. As hydrazine concentration increased from0.001% to 25%, hybridization of probes to the 9mer decreased, whereashybridization of probes to the 13mer increased with increasing hydrazineconcentration. Roughly equivalent amounts of hybridization of each probewere observed for hydrazine concentrations in the range of 0.5 to 2%.

Example 4 Immobilization of Two Aldehyde Group ContainingOligonucleotides in Parallel

As an alternative to the sequential immobilization methods described inExample 1, one approach is to mix two aldehyde group containingoligonucleotides together in solution with hydrazine beads.

Hydrazine beads were prepared as described in Example I and mixtures oftwo aldehyde group containing oligonucleotides having lengths of 25, 50or 75 nucleotides were reacted with the beads under the conditionsdescribed in Example 2. Surprisingly, the sequence of theoligonucleotides present in the mixtures did not adversely alter thefinal ratio of hybridization competent oligonucleotides immobilized onthe beads. FACS hybridization assays were run for beads synthesizedusing different mixtures containing the same first oligonucleotidesequence in the presence of different second oligonucleotide sequences.The results showed that differences in the composition of the secondsequence did not significantly alter the efficiency of immobilizationfor the first oligonucleotide. Similar analyses using mixtures 75mer and23mer oligonucleotides showed that the efficiency of 75merimmobilization to beads was not altered by differences in the sequenceof 23mers in the mixture. Conversely, the efficiency of 23merimmobilization to beads was not altered by differences in the sequenceof 75mers in the mixture.

These results indicate that equivalent amounts of different aldehydegroup containing oligonucleotides can be immobilized to hydrazine beadsusing equimolar mixtures of the two oligonucleotides independent ofoligonucleotide size or sequence.

Example 5 Synthesis of Aldehyde Group Containing Oligonucleotides

Amine-containing oligonucleotides that were still protected and attachedto CPG, were washed three times with 200 μL of acetonitrile. Followingthe wash, 50 μL of solution containing 0.1 MO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), 0.1 M 4-carboxybenzaldehyde, and 0.2 Mdiisopropylethylamine was added to the CPG and incubated for 15 min. TheCPG was then washed once with 200 μL of acetonitrile. The sequence ofadding the 50 μL reaction solution for a 15 minute reaction and washingin 200 μL was repeated three more times. The oligonucleotide wasdeprotected with ammonia for 8 hours, extracted and eluted in water

Example 6 Attachment of Oligonucleotides to Beads Using AldehydeModified Oligonucleotides

DNA Synthesis:

Oligonucleotide (oligo) was synthesized on CPG by standardphosphoamidite chemistry using ABI Expedite DNA synthesizer or aproprietary 96-well or 384-well Oligator DNA synthesizer. A formylindolephosphoramidite modifier from Link Technologies was used to introduce analdehyde residue into the 5′ end of the oligonucleotides. Afterformylindole amidite coupling, the DMT was removed using 100 μL of 3%dichloroacetic acid in dichloromethane at room temperature for 10minutes, the product was then washed with 1 mL of acetonitrile 3 times.The oligonucleotide was then cleaved off from CPG and deprotected using100 μL of 28% ammonium hydroxide in water at 55° C. for 8 hours. Aftercleavage, the solution was dried in a speedvac, and the oligonucleotidewas extracted using 100 μL of water.

Triazine Hydrazine Activated Beads Preparation:

Silica beads (1 g) were washed with 10 mL ethanol (HPLC grade). Thebeads were then suspended in 10 mL ethanol followed by addition of 50 μL(0.5%) 3-aminopropyltrimethoxysilane, and shaken for 1 hour at roomtemperature. Following silanization they were washed 5 times with 10 mLethanol and 3 times with 10 mL ether, dried at room temperature. Theamino beads (100 mg) were then suspended in acetonitrile (1 mL),followed by addition of 20 μL (0.12 mmol) DIEA(N,N-diisopropylethylamine). After a brief sonication, 10 mg (0.05 mmol)cyanuric chloride was added and the reaction mixture was shaken for 2hours at room temperature. The beads were washed 3 times with 1 mLacetonitrile.

The cyanuric activated beads were then reacted with 2% hydrazinesolution in DMF (acetonitrile can also be used) for 12 hours at roomtemperature. The beads were washed 6 times with DMF, 3 times withethanol and stored as 10% solid in ethanol until needed.

DNA Immobilization:

An aldehyde modified oligonucleotide (100 nmol in 0.1 M sodiumcitrate/citric acid, pH 5.0) was added to 2 M sodium chloride solution(100 μL). The oligonucleotide solution was added to hydrazine-activatedbeads (25 mg beads in 100 μL of 0.1 M sodium citrate/citric acid, pH5.0). The beads were shaken overnight at room temperature and thenwashed 3 times with water. FIG. 3 is a scheme showing the synthesis ofan immobilized DNA molecule using the procedure set forth above.

Throughout this application various publications and patents have beenreferenced. The disclosure of these publications in their entireties arehereby incorporated by reference in this application in order to morefully describe the state of the art to which this invention pertains.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

Example 7 Sequential Immobilization of Amino Group ContainingOligonucleotides and Aldehyde Group Containing Oligonucleotides UsingHydrazine Beads

Amino group containing oligonucleotides were reacted at roomtemperature, overnight, in aqueous buffer with cyanuric chloridemodified beads, prepared as described in Example 1. Beads were washedwith aqueous buffer and then ethanol. Following attachment of aminogroup containing oligonucleotides to the beads, aldehyde groupcontaining oligonucleotides were immobilized as described in Example I.

Activity of the beads was evaluated using a hybridization assay in whichfluorescently labeled oligonucleotide probes, having sequencescomplementary to the immobilized oligonucleotides, were hybridized tothe beads and detected in a fluorescent activated cell sorter (FACS).When sequential two-attachment chemistry is used, the hybridizationintensities for the first 23-mer, oligonucleotide 1, attached usingamino group containing oligonucleotide and second 23-mer,oligonucleotide 2, attached using aldehyde group containingoligonucleotide were affected by the hydrazine concentrations andreaction times of the hydrazine treatment. Hybridization efficiencyincreased as incubation time increased from 5 minutes to overnight. TheFACS hybridization assay was used to determine the effects of differentconcentrations of hydrazine on hybridization of probes to theoligonucleotide 1 and oligonucleotide 2. As hydrazine concentrationincreased from 0.001% to 25%, hybridization of probes to the 23-mer,oligonucleotide 1 decreased, whereas hybridization of probes to the23-mer, oligonucleotide 2 increased with increasing hydrazineconcentration. Roughly equivalent amounts of hybridization of each probewere observed for hydrazine concentrations in the range of 0.5 to 2%.

1. A compound of Formula XXVI

wherein X⁵, X⁶, X⁷, and X⁸, are each independently selected from thegroup consisting of chloro, NH—NH₂, Z-D, NH—N═CH—Z-D, NH—N═Z-D,NH—NH—Z-D and NH—Z-D, wherein, each Z is a bond or a linker; and each Dis independently a biologically active polymer; Q is a solid surface; V¹and V² are each independently selected from the group consisting of abond, SiO₃, NH, CO, NHCO, C(O)NH, (CH₂)_(p), sulfur, and oxygen, or acombination thereof; and p is an integer greater than or equal to zero.